Montero C, Llorente P
Instituto de Investigaciones Biomédicas del C.S.I.C. Arturo Duperier, Madrid, Spain.
Biochem J. 1991 Apr 15;275 ( Pt 2)(Pt 2):327-34. doi: 10.1042/bj2750327.
Adenine phosphoribosyltransferase (APRTase) and hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) have been purified from Artemia cysts and nauplii to apparent homogeneity, as determined by SDS-PAGE. The purification includes affinity chromatography on AMP-Sepharose, which binds both enzymes, and they are eluted at different 5-phospho-alpha-D-ribosyl diphosphate (PP-Rib-P) concentrations. The purified enzymes from Artemia cysts were similar to nauplii enzymes with respect to Mr in denaturing gel electrophoresis and gel filtration, pH and cation dependence and kinetic constants for substrates and inhibitors. By Sephadex G-100 filtration, the native Mr of the adenine and hypoxanthine-guanine enzymes was estimated to be Mr 28,000 and 66,000, respectively. Analysis by SDS-PAGE revealed that the APRTase was a dimer of Mr 15,000 sub-units and the HGPRTase, a tetramer of four identical Mr 19,000 sub-units. The pH profile of the HGPRTase shows two apparent buffer-independent pH optima, at 7.0 and 9.5, while the APRTase has just one, at about pH 8-9. The purine phosphoribosyltransferase activity with adenine was highest, about tenfold the HGPRTase activity with hypoxanthine and fivefold that with guanine. Both enzymes exhibited similar requirements for divalent cations, either Mg2+, Mn2+ or Zn2+, while Ca2+ is highly inhibitory. The Km values of APRTase for adenine and PP-Rib-P are 2 and 30 microM, respectively, and the Km values of HGPRTase for hypoxanthine, guanine and PP-Rib-P are less than 1, less than 1 and 15 microM, respectively. Plots of the reciprocal enzyme activities versus reciprocal concentrations of one substrate at several fixed levels of the second one yield a pattern of inhibition by guanine and hypoxanthine. Product-inhibition studies indicated that AMP is a competitive inhibitor with respect to PP-Rib-P in the APRTase reaction, while the HGPRTase shows a mixed inhibition by GMP.
已从卤虫囊肿和无节幼体中纯化出腺嘌呤磷酸核糖转移酶（APRTase）和次黄嘌呤 - 鸟嘌呤磷酸核糖转移酶（HGPRTase），经SDS - PAGE测定达到表观均一性。纯化过程包括在AMP - Sepharose上进行亲和层析，该介质可结合这两种酶，并在不同的5 - 磷酸 - α - D - 核糖基二磷酸（PP - Rib - P）浓度下洗脱。从卤虫囊肿中纯化得到的酶在变性凝胶电泳和凝胶过滤中的相对分子质量、pH和阳离子依赖性以及底物和抑制剂的动力学常数方面与无节幼体中的酶相似。通过Sephadex G - 100过滤，腺嘌呤酶和次黄嘌呤 - 鸟嘌呤酶的天然相对分子质量估计分别为28,000和66,000。SDS - PAGE分析表明，APRTase是由相对分子质量为15,000的亚基组成的二聚体，HGPRTase是由四个相对分子质量均为19,000的相同亚基组成的四聚体。HGPRTase的pH曲线显示在7.0和9.5处有两个明显的与缓冲液无关的pH最适值，而APRTase只有一个，约在pH 8 - 9。腺嘌呤的嘌呤磷酸核糖转移酶活性最高，约为HGPRTase对次黄嘌呤活性的十倍，对鸟嘌呤活性的五倍。两种酶对二价阳离子（Mg2 +、Mn2 +或Zn2 +）的需求相似，而Ca2 +具有高度抑制作用。APRTase对腺嘌呤和PP - Rib - P的Km值分别为2和30μM，HGPRTase对次黄嘌呤、鸟嘌呤和PP - Rib - P的Km值分别小于1、小于1和15μM。在第二个底物的几个固定水平下，将酶活性的倒数与一种底物浓度的倒数作图，得到鸟嘌呤和次黄嘌呤的抑制模式。产物抑制研究表明，在APRTase反应中，AMP是PP - Rib - P的竞争性抑制剂，而HGPRTase表现出GMP的混合抑制作用。
